Process of preparing and growing and distributing organisms which fix or gather atmospheric nitrogen.



UNITED s'r TEs PATENT OFFICE.

GEORGE HERBERT EARP-THOMAS, OF WELLINGTON, NEW ZEALAND.

ATMOSPHERICNITROGEN.

PROCESSOF PREPARING AND GROWING AND DISTRIBUTING ORGANISMS WHICH FIX 0R GATHER Ne. e eeeo.

. Specification of Letters Patent.

Patented April e, 1906.

To all whom it may concern:

Be it known that I, GEORGE HERBERT BARF-THOMAS, a British subject, and a resident of Wellington, in the Colony of New Zealand, a temporary resident of Toronto, in the count of York and Province of Ontario, Canada, ave invented a new and useful Process of Preparing and Growing and Distributini Organisms which Fix or Gather Atmosp ric Nitrogen; and I hereby declare that the following is a full, clear, and exact description of the same.

This discovery consists of an improved and simplified method of preparing and growing nitrogen-gathering bacteria for distribution and intensifying the virulence or infectiousness of the organisms to a degree hitherto unknown.

The process essentially consists of growing the bacteria in corked and sealed vessels by a nitrogen-fieemedium for a given period until the organisms reach a stage of-exalted virulence' The growth bein veryprofuse in the medium permits imme 'ate and direct inoculation of the seed or soil.

In carryin out this. process a nodule is obtained from t e roots of a leguminous plantsuch as alfalfa, beans, peas, clover, &c.-and Washed until the skin is thoroughly clean. The nodule is then immersed in a sterilizing solution, such as corrosive sublimate of one one-thousandth strength if the module is large or sterilized water if the nodule is small, for a period of about three minutes. The nodule is then removed from the sterilizing solution and placed on absorbent paper to dry for a period of one minute. When dried, it is seized, with a pairof sterile forceps, and the skin cut, with a sterile instrument, so that vwhen broken open it will show a clean fractured surface. A small quantity of the bacteria from the center of the nodule is then removed with a sterile'blade and placed in a drop of sterilized water in a sterilized vessel. These bacteria are then rubbed into the water until the latter turns milky.. One dro of the milky fluid is then taken on asterile p atinum loop and placed in a culture vessel containin a culture medium, and this vessel is place in an incubator heated to a temperature of 25 centigrade and kept'there from two to four days or until the bacteria colonies develop. The bacteria from these colonies are then transferred to vessels containing media to be kept when proved aspure bacteria stock culture for preparing or growing the bacteria for distribution to the users.

To prepare or grow the bacteria for distribution to the users, suitable vessels or bottles containing "speciallyprepared media free from nitrogen and of a solid non-fluid or liquid nature, but preferably solid, are em-. ployed, and in each of these vessels is placed a small quantity of the pure bacteria stock culture. These vessels are then placed" in an incubator, heated to a temperature of approximately 25 centigrade, and kept there for a period of about four days or until the bacteria colonies have developed. Owing to the lack of nitrogen, the bacteria are in a very virulent condition, which may be de? scribed as meaning that the vital properties of the bacteria are exalted to such a state that they will rapidly multiply and eagerly seek for nitrogen. In preparing the special medium fourteen grams of wood-ash are steeped in two thousand two hundred and ten cubic centimeters of clear water, heated to 212 Fahrenheit,for aperiod of one minute and then filtered clear. Intothis clear filtrate put a stiffening agent, such as twent six grams of shredded agar, and boil it till t e agar is dissolved. Then put into this solution thirty-five grams of dissolved maltose and then filter the final solution till quite clear. This filtrate is ut into bottles onethird full and sterilize for half an hour on each of three successive days. As the agar is hard to filter, another method to prepare this medium' is to heat the ash in one-quarter of the uantity of water and the agar in the other t ee-quarters and ,filter each separately before adding the maltose. When the maltose has been added and dissolved, the

final solution is filtered and sterilized, as

above described. The 'ash being of a distinctly-alkaline nature by its reactions upon the acids of th other constituents acts as a neutralizing agent and produces a neutral medium or a, i'aintly-alkaline nutrient medium.

The vessels or bottles 20f bacteria may be.

are packed in suitable cases and sent to the I user with directions stating) the quantity of seed the contents are capa le of inoculating IILI and the amount of water to be used. The

user has then only to empty the bacteria from the bottle into the quantity of water marked 1n his directions and then spray'this water on his seed or dip them into it. The seed may then be planted immediately or dried and planted in a few days, and in the former case the addition of a minute quantity of sugar and ash in the water is an advantage. This method 1s so simple that the user cannot reasonably make any mistake and is much quicker and less troublesome than other methods in which he has to develop the bacteria after receiving them, such development requiring skilled or scientific knowledge and even with the exercise of the greatest care entailingthe risk of s oiling the whole culture. With the pure cu ture grown in this method the user will have no trouble in obtaining the best results in every case. If the organisms repared by this rocess are not used before t ey ave attaine their maximum rowth and have deteriorated in virulence, t ey may be restored to active virulence by the employment of a small quantity of a liquid medium consisting of approximately fifteen hundred cubic centimetersof water, ten grams ash, and twenty-nine grams maltose, more or less. The organisms are kept growing on themethen to bring them to active virulence the neck of both bottles should be heated with a flame for a few seconds to sterilize them, and the corks removed, and a small quantity of the liquid medium poured into the organisms i on the solidmedium, and the bottle of culture recorked and placed in a warmroom from' Y gen the ottle of bacteria culture can be developed by the user as follows: Boil one int of sugar and half a pint of plain wood-as in five uarts of fresh water until .the sugar is diss o ved, then strain this through canvas or linen to remove the debris of ash, and while hot pofir into a tub containing ten gallons of water, preferably boiled and cooled. Pour a little water previously boiled and cooled into the bottle of culture and then Wash thor-' oughly all the bacteria from the bottle of culture into the tub of water.. The tub of water, which must not be more than milk-warm when the bacteria are placed in it, is then covered to prevent contamination, and preferably kept ina warm room at an even tem- 6 5 perature from two to five days until the wadium till two or three days before using, and

ter-is milky. The milky water is then mixed with from one hundred to five hundred gal- .lons of water and sprayed on the fields, which i will be well sup lied with the organisms, if the directions have been observed. The proper proportions of'sugar and ash, with dimotions for use, can be sent with each bottle of bacteria tothe users, stating the extent of ound they will fertilize after development; ut the users can increase the proportions of Sn ar, ash, and water according to their indivi ual requirements. A satisfactory method of reparing a liquid medium is as follows: Boil two and a-half gallons of clean water with two ounces of plain wood-ash for five minutes and then strain with linen and lace the clear filtrate in a clean vessel an boil again with five and onehalf'ounces of maltose or sugar till the sugar is dissolved-say from five to ten minutes then cool to milkwarm and add the bacteria to it by washing them out 30f the bottles with water previously boiled and cooled. Now cover up this solution for two days free from dust until the water is milky. A good way to hasten the growth of the bacteria at this period is to add two ounces of ammonium phosphate for forty-eight hours .and when milky dilute it with water, and then put it on the fields or seeds. The seeds must be kept out of the 95 sunlight afterino'culation. One pint of this milky solution will be strong enough to with five gallons of water. If necessary to pre are larger quantities of bacteria, this mil y water may be put into a tank of Water- Ice prepared with sugar and ash according to the correct proportions and then kept till milky and sprayed on the fields after sunset.

The chemical reactionsresulting from the affinity of the ash and sugar results, as in the IO 5 case of the ash and maltose, in the roduc- 1 tion ofa neutral medium or a faint y-alkaline nutrient medium.

In the specification and claims I have used the term distribution vessel to designate such receptacles as are suitable tocontain a nutrient medium in which the nitrogen-gathering bacteria may be transported by mail, express, or freight to distribute the bacteria from the laboratory to the purchasers, such 115 a distribution vessel being distinguished by the fact that it may contain the medium in a moist condition, whereby the bacteria may be distributed in a virulent condition as distinguished from the distribution in a dry con- 1 2o dition upon dry fibrous material, which is not anutrient medium, and upon whichthe bacteria are in ractically a spore or dormant condition, 5on1 which spore must afterward be produced or developed the virulent 12 5 bacteria-that is to say, ropagated-in something the same manner t at a plant may be said to be propagated from a seed.

In referring to the restoration of the bacteria by supplying them with a restoring me- 1 o dium, which step is an important feature of my invention, I use the term restoring to distinguish from developin as indicatm a process of reviving the moti e bacteria as istinguished from propagating such bacteria from dried spores or large rods.

Having thus fully described my invention, what I claim as new, and desire to secure by Letters Patent, is

1. The herein-described process which consists of thoroughly cleaning and sterilizing the nodule of a leguminous lant, then fracturing it while sterilized, an obtaining from it a quantity of bacteria in a bacteriologically-pure condition and mixing them with a corresponding quantity of sterilized water until a milky fluid is produced, then taking a predetermined quantity of the milky fluid and placing it in a culture vessel with a medium substantiallyfree from nitrogen, and maintaining the culture vessel at developing temperature until the bacteria colonies apear. p 2. The herein-described process which consists of thoroughly cleaning and sterilizing the nodule of a leguminous lant, then fracturing it while sterilized, an obtaining from it a quantity of bacteria in a bacteriologically-pure condition and mixing them with a corresponding quantity of sterilized water until a milky fluid is roduced, then taking a predetermined quantity of the milky fluid and placing it me culture vessel with a medium substantially free from nitrogen, and

maintaining the culture vessel at developingtemperature until the bacteria colonies appear, then transferring part of the bacteria colonies to distributing vessels containing media, and maintaining these distributing vessels at a developing tem erature until the bacteria colonies have deve oped.

3. The herein-described process which consists of thoroughly cleaning and sterilizing the nodule of a leguminous plant of the nitrogenous class, then fracturing the nodule While sterilized, and obtaining from it a quantity of bacteria in a bacteriologicallypure condition, then mixing with it a corresponding uantity of sterilized water until a milky fluid is produced, then taking a small quantity of the milky fluid and placing it in a culture vessel containing a medium and maintaining this culture vessel and its contents'at asubstantially even temperature of approximately 25 centi ade until the bacteria colonies a pear, t en transferring a small quantity 0 the bacteria colonies to distribution vessels containing media, and main taining these last-mentioned vessels at a substantially even temperature of. about 25 centigrade until the bacteria colonies have developed. i

4:. A process for preparin distributing organisms which atmospheric nitrogenconsisting rowing and x or gather of preparing and growing the organisms and maintaining them virulent in distributing'vessels.

5. A process for preparing, growing and distributing organisms which fix or gather at mospheric nitrogen consisting-of preparing and growing the organisms and maintaining them virulent in a distributing vessel by a medilim containefd therein. d

6. rocess or re aring an growing and disliibuting organisms which fix or gather atmospheric nitrogen, consisting of preparing the organisms, growing and distributing them on a moist medium of a nonnitrogenous character.

7. A process for preparing and growing and distributing organisms which fix or gather atmospheric nitrogen consisting of preparing and growing the organisms bacteriologically pure and maintaining them in distributing vessels in a substantially nonnitrogeous environment.

8. The process of distributing nitrogenathering'organisms, which consists in transerring such organisms from a laboratory stock culture to a distributing vessel substantially closed against access of air and containing a medium of a non-nitrogenous character, and transporting them in said distributing vessel.

9. The process of distributin virulent nitrogen-gathering organisms, W ich consists in transporting them in a substantially nitrogen-free environment.

10. The process of restoring non-virulent organisms contained in a distributing vessel which consists in providing a vessel with a sterilized medium, bringing the vesselsinto juxtaposition and discharging the contents of one into the other.

11. The process of restoring the virulency of non-virulent nitrogengathering organ isms, which consists in supplying medium of a non-nitrogenous character to distributing vessels containing said non-virulent organ- ISIIlS.

12. A medium for the culture of. organisms which fix or gather atmospheric nitrogen comprising ash, a ar, maltose,and water.

13.. A medium or the culture of organisms which fix or gather atmospheric nitrogen com rising fourteen grams of ash, two thousand two hundred and ten cubic centimeters of water, twenty-six grams of shredded agar,

and thirty-five grams of maltose.

14. A medium for the culture of organisms which fix or gather atmos heric nitrogen com rising fourteen grams of ash, two thousan two hundred and ten cubic centimeters of water, twenty-six grams of shredded agar, and thirty-five grams of maltose, and a restoring medium roportionally consisting of fifteen hundred cubic centimeters of water, ten

grams of ash ,and twenty-six grams of maltose.

15. A non-nitrogenous medium for pre paring and growing and distributing organisms which fix or gather atmospheric nitrogen comprising, ash, carbohydrate, and water.

16. A non-nitrogenous medium for pre' paring and owing and distributing organisms which fi; or gather atmospheric nitrogen comprising carbohydrate, water and neutralizing agent.

17. A non-nitrogenous medium for preparing and isms which or gather atmospheric nitrogen comprising ash, carbohydrate, water and a stiffenlng agent.

18. Amedium containin nitrogen-gathering bacteria in sealed distri uting vessels.

19. A medium for the culture and distribu tion of nitrogen-gathering organisms, said medium being free from nitrogen and containing the constituents of wood-ash.

20. A bacteriological ditributing-package containing virulent nitrogen-gathering bacteria in a substantially nitrogen-free environment.

21. A bacteriological distributing-package comprising'a rece tacle, a non ;nitro enous medium containe therein, a culture of nitrogen-gathering organisms carried by the meggpwing and distributing organ-' dium and means for restricting the access of air to the receptacle.

22. A bacteriological distributing-package comprising a sealed receptacle, a non-nitrogenous medium contained therein and a culture of virulent nitrogen-gathering organisms carried by the medium.

23. A bacteriological distributing-package comprising a receptacle, a non-nitrogenous medium, and a culture of virulent nitrogengathering organisms carried by the medium,

said medium containing. the constituents of Wood-ash.

24. A bacteriological distributing-packagecomprising a receptacle, a culture medium, and a culture of nitrogen-gathering organisms, said medium containin ash.

25. A bacteriological distribution-package comprising a receptacle, a culture medium and virulent nitrogen-gathering organisms in said receptacle.

Toronto, September 22, 1905 GEORGE HERBERT EAltP-THOMAS.

In presence of C. H. RIoHEs, H. L. TRIMBLE. 

